The soybean (Glycine max) belongs to the Fabaceae (Leguminosae) family. This plant family is identified by having its seed borne in a legume (pod). The soybean is thought to have originated in China. Wild types of soybeans are viny in nature, which probably is a major reason why soybeans were first introduced in the United States as a hay crop. Introductions from China, Manchuria, Korea and Japan have been important in developing varieties for the United States. Modern breeding efforts to improve the agronomic traits, such as more erect growth, reduced lodging and increased seed size, have been primarily responsible for the development of soybeans into a crop of world-wide importance. The acreage and the proportion of the crop harvested for grain has increased steadily and today soybeans are a major world commodity.
Cultivated soybean has a substantial commercial value throughout the world. Over 50 million hectares worldwide are used to produce an annual crop of soybeans in excess of 100 metric tons with an estimated value exceeding 20 billion dollars. The development of scientific methods useful in improving the quantity and quality of this crop is, therefore, of significant commercial interest.
Soybeans are widely used as a source of protein, oil, condiments and chemical feed-stock. Significant effort has been expended to improve the quality of cultivated soybean species by conventional plant breeding, and a number of major successes are recorded. The methods of conventional plant breeding have been limited, however, to the movement of genes and traits from one soybean variety to the other.
Modern biotechnological research and development has provided useful techniques for the improvement of agricultural products by plant genetic engineering. Plant genetic engineering involves the transfer of a desired gene or genes into the inheritable germ-line of crop plants such that those genes can be bred into or among the elite varieties used in modern agriculture. Gene transfer techniques allow the development of new classes of elite crop varieties with improved disease resistance, herbicide tolerance, and increased nutritional value. Various methods have been developed for transferring genes into plant tissues including high velocity microprojection, microinjection, electroporation, direct DNA uptake, and Agrobacterium-mediated gene transformation. Agrobacterium-mediated gene transformation is the most widely used gene transfer technique in plants. This technique takes advantage of the pathogenicity of the soil dwelling bacterium Agrobacterium tumefaciens. Agrobacterium tumefaciens natively has the ability to transfer a portion of its DNA, called T-DNA, into the genome of the cells of a plant to induce those cells to produce metabolites useful for the bacterium's nutrition. Agrobacterium-mediated transformation takes advantage of this concept by replacing the T-DNA of an Agrobacterium with a foreign set of genes, thus, making the bacterium a vector capable of transferring the foreign genes into the genome of the plant cell. Typically, the foreign gene construct that is transferred into the plant cell involves a specific gene of interest, which is desired to be introduced into the germline of the plant, coupled with a selectable marker that confers upon the plant cell a resistance to a chemical selection compound. Typically, the Agrobacterium-mediated gene transfer is into an undifferentiated cell cultivated in tissue culture, known as a callus cell, or the transfer is made into a differentiated plant cell from a leaf or stem, which is then induced to become an undifferentiated callus culture.
The development of a method for introducing foreign genes into soybean species greatly enhanced the range of traits which could be imparted to soybeans. In order to obtain a system for useful gene introduction into soybeans, a number of obstacles had to be overcome. These include optimization of regeneration to whole plants of the target tissue, definition of the conditions (e.g., time, bacterial concentration, and media) for the co-cultivation of the soybean cells and Agrobacterium cells, and establishing an appropriate selection protocol.
However, DNA delivery using particle bombardment, electroporation, or Agrobacterium-mediated delivery into soybean has proven to be difficult. This is due, in part, to the small number of cells that have been found to be totipotent in soybean (Trick et al. (1997) Plant Tissue Cult Biotechnol 3:9-26). Methods that use Agrobacterium tumefaciens for DNA delivery have the additional problem of overcoming any incompatibility between the soybean explant and the Agrobacterium. Two methods routinely used are an Agrobacterium-based method targeting the cotyledonary-node axillary meristems (Hinchee et al. (1988) Bio/Technology 6:915-922) and a method using particle bombardment of mature zygotic embryos (Finer and McMullen (1991) In Vitro Cell Dev Biol 27P: 175-182).
Described are methods based on somatic embryogenesis: Embryos are induced from immature soybean cotyledons by placing the explant on high levels of 2,4-D (40 mg/L) and the embryogenic tissues are subsequently proliferated on induction medium (Finer (1988) Plant Cell Rep 7:238-241) or liquid suspension culture (Finer and Nagasawa (1988) Plant Cell Tissue Organ Cult 15:125-136).
Further described are methods based on Agrobacterium-mediated transformation of zygotic immature cotyledons (Parrott et al. (1989) Plant Cell Rep 7:615-617; Yan et al. (2000) Plant Cell Rep 19:1090-1097; Ko et al. (2003) Theor Appl Genet. 107:439-447). However, in Parrott et al. the three plants produced were chimeric, from a multicellular origin, and did not transmit the transgene to the next generation. Yan et al. (2000) Plant Cell Rep 19:1090-1097 reported a low transformation frequency of 0.03%. Plant produced transmitted the transgene into the next generation, presumably due to the continuous selection of transformed primary embryos for the production of secondary embryos thereby resulting in non-chimeric plants. Recently, Ko et al. (2003) Theor Appl Genet. 107:439-447 has reported the recovery of transgenic plants at 1.7% transformation frequencies, however, the method relies on using a partially disarmed (oncogenic) Agrobacterium strain, pKYRT, with a functional TR-DNA sequence in order to stimulate embryogenesis (Ko et al. (2004) Planta 218:536-541). These methods use the immature cotyledons as the target tissue with subsequent proliferation and selection on solid medium.
Other methods for soybean transformation are based on particle bombardment trans-formation of proliferative embryogenic cultures. Fertile transgenic soybean plants have been produced using particle bombardment (Finer and McMullen (1991) In Vitro Cell Dev Biol 27P:175-182; Sato et al. (1993) Plant Cell Rep 12:408-413; Parrott et al. (1994) In Vitro Cell Dev Biol 30P:144-149; Hadi et al. (1996) Plant Cell Rep 15:500-505; Stewart et al. (1995) Plant Physiol 112:121-129; Maughan et al. (1999) In Vitro Cell Dev Biol-Plant 35:334-349). In these methods, the proliferative embryogenic cultures from both liquid and solid media are used for particle bombardment and immediate selection occurs while on solid or liquid media.
The above-described methods based on embryogenic cultures have one or more of the following disadvantages:    1. A continual supply of greenhouse grown plants are needed to supply the immature cotyledons for establishment of embryogenic cultures and induction of embryo-genesis.    2. For microprojectile bombardment, induction of somatic embryos occurs for at least 90 d on solid or liquid medium before bombardment. After bombardment, the embryos are transferred to medium with selection up to 4 weeks, or when embryos elongate. Surviving embryogenic clusters are transferred to maturation medium for a minimum of 4 weeks. The mature embryos are then desiccated for 2 to 7 days then plated onto germination medium for 3 to 4 weeks. After embryos develop shoots and roots, they are transferred to Magenta boxes for 2 to 3 weeks before transferring to greenhouse. This process takes approximately 9 months to one year.    3. For Agrobacterium infection, the immature cotyledons are used as the target material thereby decreasing the time by 3 months. However, to produce non-chimeric plants, production of secondary embryos from transgenic primary embryos is needed before desiccation of mature embryos to induce germination of plantlets.    4. Sterility with somatic embryogenesis and particle bombardment is a problem (Samoylov et al. (1998) Plant Cell Rep 18:49-54). This is mainly due to the length of time in culture (see above).    5. The induction of somatic embryos and the formation of proliferative embryogenic cultures are highly genotype-dependent (Bailey et al. (1993) In Vitro Cell Dev Biol 29P:102-108; Bailey et al. (1993) Crop Sci 34:514-519; Simmonds and Donaldson (2000) Plant Cell Rep 19:485-490).
Other methods for soybean transformation are employing the embryo axes as target tissue. Methods for particle bombardment transformation of immature embryonic axes are disclosed (McCabe et al. (1988) Bio/Technology 6:923-926; Aragao et al. (2000) Theor Appl Genet. 101:1-6). The embryos of mature, sterile seeds are excised and the apical meristem exposed by removing the primary leaves. After bombardment of the apical meristem, the explants are moved to shoot induction medium overnight and the explants are transferred to recovery plus selection medium for 2 weeks before elongated shoots begin to emerge. After 3 to 4 weeks additional shoots regenerate. A total of 5 to 7 shoots regenerate in total, and in Aragao et al. (2000), only 10% of those shoots elongated. Transformation efficiency from 0.1 to 20.1%. This group used ahas (acetohydroxyacid synthase) for selection of transgenic cells while the protocol from McCabe et al. (1988) Bio/Technology 6:923-926 no selection is applied. Agrobacterium mediated transformation of immature embryo axes is further described in US 20030046733 and U.S. Pat. No. 6,384,301 with a 1 to 3% transformation efficiency. The protocol is similar to above, but instead of bombardment, Agrobacterium is applied and a co-cultivation step included. Also, pretreatment of seeds with hormones is claimed.
Other methods related to transformation of the cotyledonary-node, e.g. by particle bombardment (U.S. Pat. No. 5,322,783). The cotyledonary node is targeted after excising the meristem from imbibed seeds, a pretreatment with cytokinins for 1 day, and a preculture on sucrose medium for an additional day. In this patent no transformed plants are presented. Presumably this method would be difficult to access the cells for particle bombardment. Transformed plants have been reported by using Agrobacterium tumefaciens infection of the cotyledonary-node (Hinchee et al. (1988) Bio/Technology 6:915-922; Zhang et al. (1999) Plant Cell Tissue Organ Cult 56:37-46; Olhoft and Somers (2001) Plant Cell Rep 20:706-711; Olhoft et al. (2003) Planta 216:723-735). Explants are prepared from 5-day-old seedlings and exposed to Agrobacterium tumefaciens. After co-cultivation, shoots are induced for 4 weeks under selection. Elongation of transformed shoots begins as early as 4 to 6 weeks on elongation medium and continues for 6 months. Transformed shoots are rooted on rooting medium for 5 to 7 days before transferring to the greenhouse.
Although some of the problems linked to the transformation of soybeans have been overcome by the methods described in the art, there is still a significant need for improvement, since all methods known so far have only a low to moderate transformation and—especially—regeneration efficiency. Although significant advances have been made in the field of Agrobacterium-mediated transformation methods, a need continues to exist for improved methods to facilitate the ease, speed and efficiency of such methods for transformation of soybean plants. Therefore, it was the objective of the present invention to provide an improved method having higher overall efficiency in the process of generation of transgenic soybean plants. This objective is solved by the present invention.